Anti-microbial potentiation with agents having plant hormone activity



United States Patent 3,251,738 ANTI-MICROBIAL POTENTIATION WITH AGENTS HAVING PLANT HORMONE ACTIVITY George H. Scherr, 50 Monee Road, Park Forest, 11]. No Drawing. Filed Mar. 8, 1963, Ser. No. 263,753 30 Claims. (Cl. 167-65) 263,754, also filed March 8, 1963, and which is now abandonded.

The term synergism-has not always been used to mean the same thing. In general, however, the term synergism has been used to refer to the activity of two agents both of which have some anti-microbial activity but which in combination display an activity greater than the sum of the two activities when each agent is used alone. Oftentimes an additive effect may be erroneously referred to as synergistic. Occasionally the term synergism has been used to refer to the combined action of two agents where one of the agents alone had little or no antimicrobial activity. The term potentiation, in my opinion, more properly refers to the augmenting of the activity of an antibiotic agent by a substance which in itself has little or no anti-microbial activity. Discussions of the principles underlying synergism and/ or potentiation have been discussed in such papers as those by Eagle and Saz, Annual Review of Microbiology, volume 9, pages 173- 226, 1955; Lacy, 8th Symposium of the Society for Gen- .eral Microbiology, 1958, Cambridge University Press;

Jawetz and Gunnison, Antibiotics and Chemotherapy, volume 2, pages 243-248, 1952; and Jawetz, Gunnison, and Coleman, Journal or Microbiology, volume 10, pages 191- 198, 1954.

The disclosure of this invention will establish that an organism subjected to a concentration of antibiotic, even if the concentration is subinhibitory, can be altered in its physiology and biochemical activity in a manner that will make it more susceptible to certain agents that have little or no detectable inhibitory efiect when used alone. Consequently, although I will use the term synergism throughout this application, it should be understood that the term synergism is being used synonymously with potentiation due to the fact that possibly both mechanisms may be acting either separately or together in the various combinations which are reported.

Thus, according to the present invention, an antimicrobial agent and a potentiating agent are contacted concurrently with the bacteria until greater antibacterial activity against said bacteria is demonstrable than the corresponding antibacterial activity demonstrable from either of said constituent members, viz., the antimicrobial agent and the potentiating agent, when used alone in equivalent amounts.

According to the present invention, it has been found that the antibiotic and anti-microbial agents, penicillin, chloramphenicol, bacitracin, polymyxin B, ristocetin, iso nicotinic acid hydramide, streptomycin, and para-aminosalicylic acid form synergistic combinations with substances or agents that have plant hormone activity. By this is meant that the agents either are plant hormones or possess activity like plane hormones. The agents possess little or no measurable antibiotic activity in themselves. Such agents, however, do not form synergistic combinations with every antibiotic under discussion. This, as well as the synergistic activity found, will be clear from the experimental findings infra.

EXPERIMENT A The basic procedure utilized was the strip-gradient plate technique (see Bact. Proc. 1956, p. 30, and Antibiotics Annual, 19561957, New York, Medical Encyclopedia, Inc., 1957, pp. 906-917). Gradient plates were prepared with medium No. 3 of Grove and Randall, Assay Methods of Antibiotics: a Laboratory Manual, New York, Medical Encyclopedia, Inc., 1955, to which agar was added. For the more diflusible anti-bacterial substances, routine laboratory agar-agar was used at 1.5 percent concentration. For the large molecule antibiotics, 0.75 percent Ionagar No. 2 agar was used, since it permits better diffusion than conventional agars (Antib. & Chemo. 8: 599, 1958). Each layer of the gradient contained 12.5 ml. of medium, with the antibiotic incorporated in the upper layer. These plates were overlaid with 3.5 ml. of seeded agar.

The test organisms used in these studies were Staphylococcus aureus 209 (ATCC 653 SP) and Sarcina lutea (ATCC 5341). Plates were incubated for 18 hours at 35 C. before being read.

The antibiotic solutions were prepared in accordance with the instructions of Grove and Randall, supra. The antibiotics were dissolved in the upper layers of the gradient plates at the following concentrations: S. lutea penicillin, 0.03 unit; chloramphenicol, 0.30 g; Staphylococcus penicillin, 0.4 unit; chlorarnphenicol, 2.0 g; erythromycin, 0.25 g; bacitracin, pg; polymyxin B, g.; ristocetin, 5 g; oxytetracycline, 0.20 g; chlortetracycline, 0.10 p..; tetracycline, 0.1 g.

All agents were run against the test organisms on gradient plates without antibiotics added.

Synergism or antagonism is recorded as a fraction in which the numerator is the distance in mm., representing the limiting concentrations of the agent in the strip,which w is active for any particular concentration of the antibiotic, and in which the denominator is the distance in onate ions were responsible for reaction with agents having these radicals.

Table I following summarizes the data for interaction between the agents tested and penicillin and chloramphenicol, with S. lutea as the test organism. For the tests the agents were diluted or dissolved in water for application to the test plates, unless otherwise indicated. When other diluents, e.g., ethanol, were used, they were tested independently as potentiating agents and were found to have essentially no activity as such.

Plates on Table I STRIP-GRADIENT PLATE RESULTS WITH s. LUTEA 1 Antibacterial agent in gradient Agent on strip 0 1000 ug/strip None Penicillin Chloramphem'col Kinetin Neg Furfuryl kinetin Neg 1O Indole-3-acetic acid Neg 2,4,6-trichlorophenoxy- S. /10- Neg acetic acid. Na hthalene acetic acid Neg S. /10- Neg 01,0: -Dichloropropionic acid- Neg Tr. S- Neg. a-Phencxypropionic acid Neg A. /10 Neg. Indole-3-propionic acid"--- Neg r. A" Tr. S a(2,4-dichlorophenoxy) Neg Tr. S Neg 15 propionic acid. Sodium acetate Neg Tr. A Neg. Sodium propionate Neg Neg Neg.

penicillin against S. lutea, with penicillin, chloramphenicol, erythromycin, bacitracin, polymyxin B, and ristocetin against Staph. aureus. It will be shown infra that it also showed frank synergism with isoniazid and streptomycin against Mycobacterium tuberculosis. Indole-3-propionic acid, alpha (2,4 dichlorophenoxy)propionic acid and alpha-(2,4,5-trichlorophenoxy)propionic acid are also shown to be very active synergists.

EXPERIMENT B The strip-gradient plate method, the basic procedures utilized, and the agents tested were essentially those. of Experiment A supra. The antimycobacterial agents were prepared in solutions of phosphate buifer No. 1 of Grove and Randall and dilutions made with the same solution. The test organism was Mycobacterium tuberculosis 607, an attenuated, fast-growing strain reputed to be the Koch strain. Stock suspensions of the test organism were prepared by growing it in Medium N0. 3 of Grove and Randall for 72 hours at C. with frequent shaking to assure a farily homogeneous suspension. This suspension was kept in the refrigerator for up to 30 days, and a 0.1 ml. volume was used to inoculate 3.5 ml. of seed agar used in preparing the gradient plates. The agents Table II STRIP-GRADIENT PLATE RESULTS WITH STAPH. AURES 209 1 T etracycline Indole3-propionic acid a(2,4-dichlorophenoxy) propionic acid Agent on strip, 1,000 ug/strip None Oxytetracycline Chlortet- Penicillin Ohlorampehnicol racycline Kinetin 2 T1. Inh S. 15/l0 Furluryl kinetin z Tr. Inh S. 10/l0 Indo1e-3-acetie acid N Ne 2,4,6-trichlorophenoxyacetic acid Neg Neg I Naphthalene acetic acid N92 N00 a(2, ia5-trichlorophenoxy) propiom'c aci Sodium acetate Ne Ne Neg. Sodium propinn N N Neg.

1 Neg. no activity; fIr. trace of activity; Inh. inhibition of growth; Stim. stimulation of growth; S. synergism; A. antagonism.

2 Five hundred ig/strip.

Table III summarizes the data for the interaction between the agents tested and the four antibiotics: erythromycin, 'bactracin, polymyxin B, and ristocetin, using Staphylococcus as the test organism.

were tested against the testorganism alone. Because pyridine-3-sulfonic acid is inhibitory to the 607 strain, and also because pyradine-B-sulfonic acid and isonicotinicacid-hydrazide (isoniazid) are both antagonist of niacin,

Table III STRIP-GRADIENT PLATE RESULTS \V ITH STAPH. AUREUS 209 1 Agent on strip, 1,000 g/strip Antibacterial agent in gradient Erythromycin B acitracin Poly-myxin B Kinetin 2 Furfuryl kinetin 2 IndoleB-acetic acid. 2,4,fi-trichlorophenoxyacetic acid Naphthalene acetic acid a-Phenoxypropionic acid Indole-B-propionic acid a(2,4-dichlorophcnoxy) propionic acid Alpha (2,4,5-trichlor0phenoxy) propionic acid Sodium acetate Sodium propionate 1 For code, see Table I. 2 Five hundred g. of agent used per strip.

It is most significant that a few of the compounds showed consistent synergistic activity with a variety of antibiotics and organisms studied. For example, 2,4,6-

it was decided to add pyridine-3-sulfonic add to the series of anti-mycobacterial agents tested; the comparative inhibitory efiect of the compound is depicted .in

trichlorophenoxyacetic acid showed frank synergism with Table IV.

I'able IV COMPARATIVE ACTIVITY OF PYRIDINE-3-SULFONIC ACID ISONIAZID AND STREPTOMYCIN BY WEIGHT USING Mycobacterium Tuberculosis 607 STRAIN ]12.7 mm. discs med] 2,4,6-trichlorophenoxyaceti-c acid and alpha-(2,4,5-trichlorophenoxy) propionic acid are plant growth hormones 5 and might well be acting as such on the microorganisms Diamete f 0 es inhibfi when certain intracellular mechanisms become susceptible concentration I 2 n 1 on to such action concomitant with exposure to antiobiotics. indisc gm.

15mm Strepto- Pyridine-3- c mycin sulionic acid Toxicity studies with a number of the agents tested 1,000 70 55 so in Experiments A and B were performed. Single doses 28 g; 3 intraperitoneal inoculations made in 25 gm. albino mice 1 16 13 indicated that the toxicity in mg/kgm. of body weight was 250 for a-(2,4,5-trich1orophenoxy) propionic acid 15 and i reater han 1 Unless otherwise indicated, the agents tested for syneracid g t 500 for mchlorophenoxyacenc gistic activity were pipetted on the strip in water, in f 2 4 v a volume of 0.1 ml. containing 1 mg. The anti-mycobac- E g fga g gg gg Pmplomc and (a TPP) terial agents were added to the upper layers of gradient plates at the following concentrations: 20 M i l d h d Agent: 'g 'mlupper layer The strain of beta-hemolytic Streptococcus used in I or gm lent P ates 2 2, the following experiments was built up in pathogenicity, psomazl Ii O'0 and would kill Swiss mice. The organism was always f q Cy g am 25 harvested from frozen dead mice which had previously "t""i& been infected by the intravenous route. Such thawed yn (mm acl u mice were autopsied and fragments from .the liver, spleen AH plates were incubated 72 hours at 35 9 C. and read or kidney, or a loopful of hearts blood, were streaked as in Experiment A. on tryptose blood agar plates and incubated for 24 hours Table V summarizes the data for interaction between at 37 C. Beta hemolyt-ic colonies were picked and transthe agents tested and M. tuberculosis 607 with four antiferred to 5 ml. of Todd-Hewitt broth in a test tube. Such mycobacterial substances. a culture, after incubation for 18 hours, could be quick- Synergism or antagonsim, when present, was designated ly frozen and stored for use at a later date if more conhere as frank synergism or antagonism; otherwise such venient. The Todd-Hewitt cultures were used as an activity was designated as trace. 7 inooulum in 0.1 ml. volumes for tryptose blood agar v Table V STRIP-GRADIENT PLATE RESULTS WITH Mycobacterz'um Tuberculosis 607 1 Anti-mycobacterial agent in gradient Agent on strip, 1,000 lg/strip None Isoniazid p-Arnino salioylic Pyridine-3 sulionic Streptomycin acid acid Kinetin Nee Tr. A- Nee Nee Tr. A.

Furfuryl kinetin Nee Tr. A- Nee Nee Tr. S.

Indole-3-acetic acid Nee Nee Nee Nee Tr. A.

2,4,Strichlorophenoxyacetic acid. Neg S. 5/30 Tr. S Neg S. 10/5 Naphthalene acetic acid. Ne Tr. S. and A- N Tr. A- Neg.

a,a-Dichloropropionic acid Nee Tr. A Nee Tr. S. Tr. A.

a-Phenoxypropionic acid- N Tr. S- Nee Neg- Tr. S.

Indole-3-propionic acid. Tr. A... Tr. S- 840/20 Tr. S.

a (gi i-dichlorophenoxy) propionic S. 10/20. Tr. S- Tr. A- Tr. S.

a(2 ,,5-trichlorophenoxy) propiom'c S. 10/30. Tr. S. Tr. A S. 30/15 So rfi ur n acetate Nee Nee Nee Nee Neg Sodium propionate- Nee Nee Nee Nee Neg 1 See Table I for legends. 2 Only 500 g. on these strips. V

The agents that showed frank synergism with strepomycin were:

(1) a-(2,4,5-trichlorophenoxy) propionic acid (2) 2,4,6-trichlorophenoxyacetic acid slants contained in large screw cap tubes and incubated for 18 hours at 37 C. Three ml. of sterile saline was added to each of these slants, the organisms'were gently washed off the slants with a sterile loop, and pooled suspensions were centrifuged at 2000-5000 rpm. for 15 minutes. The supernatant was decanted, the cells were washed a second time, and then they were transferred with saline to a Kolmer centrifuge tube to which saline was added to a fri-al volume of 10 ml. The packed cell volume was then determined so that the final suspension of organisms would result in 10 mg. of net weight of organisms per ml. of suspension; this produced 1 mg. of wet weight per 0.1 ml. of suspension, which constituted the I.V. infecting dose for mice.

Approximately 20 .gm. Swiss rnice,-whicl1 had been separated according to sex, were infected with the above suspension of cells.

In the testing of a-TPP, the animals to be treated by the intraperitoneal route were injected with either penicilbeenaddedil mg. of oc-TPP.

7 lin or the mixture of penicillin and the potentiating agent. Treatment was instituted within 24 hours after infection. If two treatment doses were administered, they were separated ,by a 48 hour period. Mice treated by the intramuscular route received .05 ml. of the antibiotic or the antibiotic and the synergist mixture.

'Ihere different experiments were performed as follows: 2 doses of penicillin of 50 units each injected I.P.; a single dose of 25 units of penicillin given by the intramuscular route; 2 doses of penicillin of 10 units each given by the intramuscular route., The doses of penicillin used here were deliberately chosen to be very close to .the minimum necessary to elicit a small increase in survivors.

The above treatment schedules were also duplicated using the same concentrations of penicillin to which had The effectiveness of the treatment schedules was assessed by counting the survivors days after infection. The results of these experiments are outlined in Table VI.

an effect on the growthof micro-organisms, it was determined if this potentiating agent also affected the rate of cell multiplication of the Streptococcus. If it were increasing the rate of multiplication, its potentiating activity might be explained by a possible increased lability of the faster multiplying Streptococcus to penicillin. However, direct cell counts, using a hemocytometer, on cultures grown in Todd-Hewitt broth, with and without 100 mcg. per ml. of the agent, showed essentially identical growth curves for 51 hours of observation.

It is demonstrated that compounds having little or no anti-microbial activity when used along acted as synergists when used in combination with antibiotics. gistic combination comprising an antibiotic and one of the synergistic agents are readily prepared by combining the materials by simple mixing or with carriers, stabilizing agents and the like. Tablets, capsules, solutions and suspensions of such compositions can be readily prepared by those skilled in the art.

Animal experiments were also performed using the Table VI POTENTIATING EFFECT OF a(2,4,5-TRICHLQROPHENOXY) PROPIONIC ACID FOR PENICILLIN ON STREPTOCOOCUS-INFECTED MICE [Number mice surviving 5 days after infection/total infected] PART 1A Infected, treated LP. Infected, treated as Infected Percent with 2 doses of 50 Percent in previous group Percent Sex untreated surviv- LU. penicillin each survivbut with 1 mg. posurviving 48 hours apart ing tentiating agent ing PART 13 Infected, treated as Infected Percent Infected, treated I.M. Percent in previous group Percent Sex untreated survivwith one dose of .survivbut with 1 mg. posurviving LU. penicillin ing tentiating agent ing added Total- 4/27 (14. 8) 8/29 (27. 6) 17/29 (58. 6)

PART 10 Percent Infected, treated LM. Percent Infected, treated as Percent Infected survivwith 2 doses of 10 survivin previous group but surviv- Sex untreated ing LU. each, 48 hours ing with 1 mg. potentiaing apart ting agent added Total. 4/27 (14. 8) 9/30 16/30 (53. 3)

It will be noted that, in general, the dosage of penicillin increased by approximately two-fold the percent survivors as compared to the infected untreated animals. In every single case, the addition of 1 mg. of a-TP P served still further to significantly increase the percent survivors; this can be seen in parts 1a, 1b, and 1c.

The increase in the percent survival, which can be attributed to the potentiating action of the. agent, was in every case approximately double that found when penicillin was used alone. From similar experiments. it was determined that the potentiating agent-used alone, either at this dose level of 1 mg. or at lesser or greater dose levels, hadno effect in altering the course of survival of infected animals. Furthermore, the potentiating agent when used alone on normal uninfected mice did not appear toxic at the dose schedule utilized here.

' Since the potentiating agent is a plant hormone and a number of plant hormones have been shown to have infectious agent Klebsiella pneumoniae. Swiss mice, of

20 gram weight, which had been separated according to sex, were infected I.P. with approximately 48,000 cells of a culture of Klebsiella pneumoniae. Treatment with 10 mcg. streptomycin and/ or 250 mcg. .of a-TPPor 250 mcg. 2,4,6-trich'lorophenol (TCP) was effected by a single dose intramuscular injection with the agents dissolved in 0.05 ml. of phosphate buffer.

All treatment was instituted within 24 hours after infection but not earlier than 4 hours post infection. Table VII outlines the results with a-TPPand TCP. It will be noted in Table VII that there are differences between the sexes especially evident in the surviving mice which had been'treated with the streptomycin plus the 2,4,6-trichlorophenol. It is clear that, treatment solely with the alpha-(2,4,S-trichlorophenoxy)propionic acid or 'the 2,4,6-t'richlorophenol did in no way favorably alter the course of mortality of these Klebsiella infected mice.

The syner- Table VII POTENTIATING EFFECT OF a-(2,4,5TRICHLO ROPHENOXY) PROPIONIC ACID (a-TPP) AND 2,4,6-TRICHLO ROPHENOL (TOP) FOR STREPTOMYCIN IN THE TREATMENT OF MICE INFECTED WITH K. PNEUMONIAE Number surviving mice Infected, treated Infected, treated Infected, treated Infected, treated Infected, treated Days post infection Infected, untreated with streptomycin with streptomycin with streptomycin with a-TPP with TCP and a-TPP and a-TCP M F M F M F M F M F M F Percent survival at 5 ays 33. 3 60 46 80 73 46 87 20 33. 3 26 33. 3 Overall percent survival at 5 days--- 26. 7 53. 3 76. 7 66. 7 26. 7 30. 0

Table VIII STRIP GRADIENT PLATE RESULTS WITH SARC'INA LUTEA 1 Antibacterial agent in gradient Agent on strip, 1 000 gJstrip None Penicillin Chloramphenicol p-ggl orlciphengxyacetic asid... lltlfeg. 2 1c orop enoxy ace ic r. eg. EXPERIMENT D Strip-gradient plates were used to test the potentiating ;5? T A activity of a number of agents against S. lutea, D p xy et Staphylococcus aureus No. 209, and ru be rculosis N0. 2 4 fsfjf i m h y acetic S 607, llSlIlg the antimicrobial agents penlclllin, chlorarn- 2 39 i m h f S 10/10 N phenicol, erythromycin, tetracycline, chlortetracycline, r 6 enoxyace r oxytetracycline, 'ristocetin, polymyxin B, bacitracin, g i gggg p y s. 20/ 0 lsocrlnazid, p-ammosalrcylic acid, pyndme-3-sulfomc acid, p tg hlorophenoxyacetic s.20/15 s. 5/65. an streptomycin. a t

D hi 0 re d... T .S Ne.

T ables VIII, IX, and X present the data for the orgfi ifiggfiggaii 3. 71 1630/10 Neg. gamsm S. lutea, S. aureus, and M. tuberculoszs N0. 607, KE -P P Y) PIOPIOHIC Neg respectively. 3(P-ch lorophenoxy) propionic s. 2o 5 Neg.

ac Alpha(2,4-dich1cr0phen0xy) Tr. S Neg. propionic acid.

2,4,5-trichlorophenoxypro- Tr. S Neg pionic acid (Alpha). 3(2,4,5'Irichlorophenoxy) S. 30/15- Neg 7O propionic acid.

Indolei-propionic acid Tr. A Tr. S Alpha-naphthaleneacetic acid. S. 15/10.-- Indole-B-acetic acid N S. 5 Neg. 2,4,6-trichlorophenol Tr. A. and S S. 5/65.

75 1 See Table I for code designations.

Table IX STRIP-GRADIENT PLATE RESULTS WITH STAPH. AUREUS 209 Agent on strip 1000 Hg/strip Antibacterial agent ingredient Penicillin Chloramphenieol Erythromycin Tetracycline p-Chlorophenoxyacetic d 2,4 dichlorphenoxyacetic acid 2,5-dichlorophenoxyacetic acid- 3,4-dichlorophenoxyacetic acid- 2,4,5-trichloropbenoxyacetic acid. 2,4,B-trichlorophenoxyacetic acid 2,3,4,6-tetrachlorophenoxyacetic acid. Pentachlorophenoxyacetic acid a-Phenoxypropionic acid 2(p-chlorophenoxy) propionic acid--. 3(p-chlorophenoxy) propionic acid a(2, iadichlorophenoxy) propionic ac 2,4,fi-trichlorophenoxypropionic acid' S. 12/20--. S. 10/

acid (alpha). 3(2,4 ,5-trichlorophenoxy) propionic A. 5/50 S. 20/

3C Indole-3-propionic acid Tr. Inh.-- N Tr. S Tr. S Tr. S. alpha-Naphthalene acetic acid Neg A. /5-- Neg.-. S. 7/10 A. /15. Indole-B-acetic acid" N Neg.. Neg S. 10/ A. 10/5. 2,4,6-trichlorophenol 10 rnm.'Inh N Tr. S S. 15/ S. 25/30 p-Chlorophenoxyacetic acid S. 10/15 Nee N Neg Tr. S. 2,4-dichlorophenoxyacetic acid Neg Tr. A-.- Neg.-. Neg" Neg. 2,5-dichlorophenoxyacetic acid Tr. A Tr. A Neg N Neg. 3,4-dichlorophenoxyacetic acid S. 15/5 Tr. A S. 15/ S. 5/35 S. 10/10 2,4,5-trichlorophenoxyacetic acid S. 15/ T1. A A. /5 S. 5/60 A. 10/10 2,4,6-trichlorophenoxyacetic acid Tr. A. and S- NP! S. /15 S. 20/35 S. 22/10. 2,3,4,6-tetrachlorophenoxyacetic acid- A. 5/40 Tr. A S. 5/ A. 5/30 A. 15/20. Pentachlorophenoxy-acetic acid A. 5/30 S. 10/40 e2 Neg A. 10/10 a-Phenoxypropionic acid S. 10/10 Neg Tr. A. and S Neg. Neg. 2(P-chlorophenoxy) propionic acid Neg Neg" eg Tr. S Neg. 3(P-chlorophenoxy) propionic acid--- S. 10/ Neg--. S. 25/10 Tr. S Tr. S. a(2,eadichlorophenoxy) propionic r. S Tr. A. and S Tr. S S. 5/35 Tr. S.

acr 2,4,S-trichlorophenosy'prupionic acid Tr. S Tr. S. S. 25/ S. 10/60 S. 10/20.

acid (alpha 3(2,4 ,(.i5-trichlorophenoxy) propionic S. 10/20 S. 10/15.... N g N S. 10/20.

3.01 Indole-3-propionic acid Tr. S S. 15/15 Tr. A Tr. A S. 5/10. alpha-Naphthalene acetic acid S. 10/15 Neg.. Tr. A Tr. S Neg. Indole-3-acetic acid S. 20/10 Neg. A. 7/5.-- N Tr. A. 2,4,6-trichl0r0phenol. S. 25/3 S. S. 20/35 Tr. S S. 10/40 1 See Table I for legends.

Table X STRIP-GRADIENT PLATE RESULTS WITH M. TUBERCULOSIS NO. 607 1 Antibacterial agent in gradient Agent on Strip 1,000 ig/strip None lsonlazld p -An 1.ino- Pyridine- Streptomycin salicylic acid 3-su1fonic acid p-Chlorophenoxyacetic acid. N Ne N92 N99 Neg 2,4-dichlorophenoxyacetic acid- Tr. Inh N N Tr. A-- Tr. A. 2,5-dichlorophenoxyacetic acid- Tr. Inh. Tr. A.- N A. 10/10 S. 10/5. 3,4-dichlorophenoxyacetic acid. N Tr. A" N A. 15/l0. Tr. S. 2,4,5-trichlorophenoxyacetic acid. Tr. Inh S. 10/10- N A. 10/5 Tr, S. 2,4,fi-trichloropbenoxyacetic acid".-. Neg S. 5/30- Tr. S- Nev S, 1015. 2,3,4,6-tetrachlorophenoxyacetic acid- Tr. Inh. Tr. S- Tr. S m1 T s Pentachlorophenoxyacetic acid 10 mm. Inh A. 5/25- A. 5/40. A. 5140. S. 10/10. a,a-Dichioropropionic cid Non Tr. A N Tr. S- r. A. a-Phenoxypropionic acid- N Tr. S- N N Tr. S. 2(p-chlorophenoxy) propionic acid.-- Neg S. 25/15- Neg Npp TL 3 3(p-chlorophenoxy) propionic acid-" N Tr. S- N A. 25/10- Tr. A. a-(2, 4-dichl0!0Ph8l1OXY) propiomc Neg S. 10/20- Tr. S. Tr. A Tr, s,

ac 2,4,etficblorophenoxypropionic acid Neg S. 10/30- Tr. S- Tr. A- S 30/15,

p a 3(2A g-trichlorophenoxy) propionic 10 mm. Inh- A. 515.. N S. 5/l5- Tr. S.

aci Indole-3-propionic acid- N Tr. A" Tr. S- S. 10/30- Tr. S. alpha-Naphthalene acetic acid Ne Tr. A. and S N Tr. Neg. Indole-3-acetic acid" N N N N Tr. A. 2,4,6-trichlorophenol 10 mm. Inh S. 30/25- S. 5/40. S. 5/50, s, 15/10,

1 See Table I for code legends.

'ristocetin, and bacitracin against S. aureus, and with isoniazid, p-aminosalicyclic acid, pyridine 3 sulfonic acid, and streptomycin against M. tuberculosis No. 607. The related antibiotics, tetracycline, oxytetracycline and chlortetracycline shall also be designated as tetracycline type antibiotics.

Certain of the antibiotics were particularly susceptible to potentiation by these agents. Thus, the anti-microbial activity of erythromycin was enhanced by many of the agents tested. A number of agents have been disclosed which are plant hormones or which are compounds that possess plant hormone-like activity. In addition to the specific agents disclosed, it is intended that conventional derivatives of such agents beincluded with the disclosure which have the plant hormone activity.

Another agent useful together with antimicrobials as a potentiating agent therefor is desyl chloride. This compound is particularly useful as a potentiating agent forpenicillin, especially against streptococci such as betahemolytic streptococci, and for streptomycin, especially against Klebsz'ella pneumoniae, as was originally disclosed in my copending application Serial No. 263,754, also filed March 8, 1963 (which is also a continuation-impart of my two earlier-filed applications Serial Nos. 54,842 and 846,265, identified in column 1 hereof), and in my application Serial No. 54,842.

Various changes and modifications of the invention can be made and, to the extent that such variations incorporate the spirit of this invention, they are intended to be included within the scope'of the appended claims.

I claim:

1. A method of producing augmented antimicrobial activity against pathogenic bacteria which .are pathogenic to man and other vertebrates, including strains of Streptococcus, Staphylococcus aureus, Sarcina lutea, Mycobacterium tuberculosis, and Klebsiella pneumoniae, which comprises concurrently contacting said bacteria with a member selected from a first group and .a member selected from a second group until greater antimicrobial activity against said bacteria is demonstrable than the corresponding antibacterial activity demonstrable from said constituent members when used alone in equivalent amounts, said first group consisting of the following members:

and said second group consisting of the following members:

(a) indole-3-acetic acid (b) naphthalene acetic acid (c) alpha-naphthalene acetic acid (d) pentachlorophenoxyacetic acid (e) 2,3,4,6-tetrachlorophenoxyacetic acid (f) 2,4,6-trichlorophenoxyacetic acid (g) 2,4,S-trichlorophenoxyacetic acid (h) 3,4-dichlorophenoxyacetic acid (i) 2,5-dichlorophenoxyacetic acid (j) 2,4-dichlorophenoXyacetic acid (k) p-chlorophenoxyacetic acid (1) indole-3-propionic acid (111) 2,2-dichloropropionic acid (11) 3-(2,4,5-trichlorophenoxy) propionic acid (o) alpha-(2,4,5-trichlorophenoxy) propionic acid (p) alpha-(2,4-dichlorophenoxy) propionic acid (q) 2-(p-chlorophenoxy) propionic acid (r) 3-(p-chlorophenoxy) propionic acid (s) alpha-phenoxypropionic acid (t) 2,4,6-trichlorophenol (u) kinetin (v) furfuryl kinetin, and

(w) desyl chloride 2. The method of claim 1, wherein the member selected from the first group is present in an inhibitory concentration.

, 3. The method of claim 1, wherein the member selected from the first group is present in a sub-inhibitory concentration.

4. The method of claim 1, wherein the member 'from the first group is oxytetracycline and wherein the member from the second group is pentachlorophenoxyacetic acid.

5. The method of claim 1, wherein the member from the first group is oxytetracycline and wherein the member from the second group is 3-(2,4,5-trichlorophenoxy)- propionic acid.

6. The method of claim 1, wherein the member from the first group is oxytetracycline and wherein the member from the second group is 2,4,6-trichloropheno1.

7. The method of claim 1, wherein the member from the first group is oxytetracycline and wherein the member from the second group is kinetin.

8. The method of claim 1, wherein the member from the first group is oxytetracycline and wherein the member from the second group is furfuryl kinetin.

9. The method of claim 1, wherein the member from the first group is tetracycline and wherein the member from said second group is pentachlorophenoxyacetic acid.

10. The method of claim 1, wherein the member from the first group is tetracycline and wherein the member from said second group is 2,4,S-trichlorophenoxyacetic acid.

11. The method of claim 1, wherein the member from the first group is tetracycline and wherein the member from said second group is 2,2-dichloropropionic acid.

12. The method of claim 1, wherein the member from the first group is tetracycline and wherein the member from said second group is 3- (2,4,5-trichlorophenoxy) propionic acid.

13. The method of claim 1, wherein the member from the first group is tetracycline and wherein the member from said second, group is alpha-(2,4,5-trichlorophenoxy) propionic acid.

14. The method of claim 1, wherein the member from the first group in tetracycline and wherein the member from said second group is alpha-(2,4-dichlorophenoxy) propionic acid.

15. The method of claim 1, wherein the member from the first group is tetracycline and wherein the member from said second group is 2,4,6-trichlorophenol.

16. The method of claim 1, wherein the member from the first group is tetracycline and wherein the member from said second group is kinetin.

17. The method of claim 1, wherein the member from the first group is tetracycline and wherein the member from said second group is furfuryl kinetin.

18. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is indole-3-acetic acid.

19. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the mernber from the second group is naphthalene acetic acid.

20. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is 2,4,S-trichlorophcnoxyacetic acid.

21. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is'3,4-dichlorophenoxyaoetic acid.

22. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is p-chlorophenoxyaoetic acid.

23. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is indole-3-propionic acid.

24. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is 2,2-dichloropropionic acid.

25. The methodof claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is 3-(2,4,5-trichlorophenoxy) propionic acid.

26. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is 3-(p-chlorophenoxy)propicnic acid.

27. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the member from the second group is alpha-phenoxypropionic acid.

28. The method of claim 1, wherein the memberfrom the first group is chlortetracycline and wherein the member from the second group is 2,4,6-trichlorophenol.

29. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the memher from the second group is kinetin.

.30. The method of claim 1, wherein the member from the first group is chlortetracycline and wherein the mem ber from the second group is furfuryl kinetin.

References Cited by the Examiner Bechtle and Scherr: Studies on Synergists for Antimicrobial Agents,, AntimycobacterialAgents, published in Antibiotics and Chemotherapy, vol. 9, No. 12, pp. 715- 721, December 1959.

Chem. Abstracts 41: 10166; 6601G (1947). Chem. Abstracts 42: 5954i-5955c (1948). Chem. Abstracts 43: 21846; 2271e-2273h (1949). Chem. Abstracts 45: 9128-1) (1951). Chem Abstracts 47: 4333i; 7038a (1953). Chem. Abstracts 49: 40881 5596b (1955 Chem Abstracts 51: 8896B (1957). Chem Abstracts 52: 4099B; 12090i-12091a; 2040413 (1958).

Chem. Abstracts 54: 10214e (May 25, 1960).

Davis et al.: Inducing Disease Resistance With Plant Growth Regulators, Phytopath 43: :137- 140 (1953).

Goodman et al.: The Efiects of Indo1e-3-(Acetic Acid,

LEWIS GO'ITS, Primary Examiner. FRANK CACCIAPAGLLA, JR., Examiner. 

1. A METHOD OF PRODUCING AUGMENTED ANTIMICROBIAL ACTIVITY AGAINST PATHOGENIC BACTERIA WHICH ARE PATHOGENIC TO MAN AND OTHER VETEBRATES, INCLUDING STRAINS OF STREPTOCOCCU, STAPHYLOCOCCUS, AURCUS, SARCINA LUTEA, MYCOBACTERIUM TUBERCULOSIS, AND KLEBSIELLA PNEUMONIAE, WHICH COMPRISES CONCURRENTLY CONTACTING SAID BACTERIA WITH A MEMBER SELECTED FROM A FIRST GROUP AND A MEMBER SELECTED FROM A SECOND GROUP UNTIL GREATER ANTIMICROBIAL ACTIVITY AGAINST SAID BACTERIA IS DEMONSTRABLE THAN THE CORRESPONDING ANTIBACTIAL ACTIVITY DEMONSTRABLE FROM SAID CONSTITUENT MEMBERS WHEN USED ALONG IN EQUIVALENT AMOUNTS, SAID FIRST GROUP CONSISTING OF THE FOLLOWING MEMBERS: 